1. Field of Invention
The present invention relates to a method for the synthesis of chromium complexes of formula [Cr3O(carboxylate)6 (H2O)3]+, wherein the synthesis uses simple and inexpensive starting materials and no organic solvents, and can be performed while generating little or no toxic by-products.
2. Discussion of the Background
In the late 1950s and 1960s, rats fed a chromium-deficient diet were found to possess an apparent decreased ability to repress blood glucose concentrations, while chromic ions were shown to increase the efficiency of insulin action in rat epididymal tissue [1-5]. Since these observations, a search has been underway to identify the biologically active form of chromium, that is, the biomolecule which naturally binds chromium (III) and possesses an intrinsic function associated with insulin action in mammals [6-8]. The average American diet contains only about 30 μg Cr per day [9, 10], which has resulted in the development of chromium-containing dietary supplements. Such materials also have potential as insulin-potentiating therapeutics which could possibly see use in the treatment of diabetes and related conditions [11]. Determining the structure, function, and mode of action of the biologically active form of chromium could greatly aid in the rational design of such potential therapeutics.
The first chromium-containing species proposed to be biologically active was glucose tolerance factor(GTF)[1,12]. GTF was first isolated from acid-hydrolyzed porcine kidney powder, although a similar, if not identical, material was subsequently isolated from yeast[1,13]. Currently the term GTF is usually understood to refer to only the material isolated from yeast. GTF is absorbed better than simple chromic salts and potentiates insulin action in rat epididymal tissue or isolated rat adipocytes [14]. However, kinetics studies indicate that GTF does not intrinsically possess biological activity [15]; additionally, the material is apparently a byproduct of the acid hydrolysis step used in its purification [16].
GTF was proposed to be composed of chromic ion, nicotinic acid, and the amino acids glycine, glutamic acid and cysteine [13]. While these results have not been reproducible in some laboratories [17-21], this report stimulated an intense interest in the synthesis of chromic-nicotinate complexes [22-25], some of which have been patented as nutritional supplements. The proposed identification of nicotinic acid (2-carboxypyridine) also stimulated investigations of complexes of chromium(III) with the related pyridine carboxylic acids picolinic acid (2-carboxypyridine) and isonicotinic acid (4-carboxypyridine) [26-28]. As a result chromium(III) tris(picolinate), Cr(pic)3, has become a very popular nutritional supplement and is being tested as a therapeutic for the treatment of symptoms of adult-onset diabetes. It is available over-the-counter in the form of pills, chewing gums, sport drinks, and nutrition bars. Cr(pic)3 has been proposed to be the biologically active form of chromium [29]. This is, however, extremely doubtful given the chemistry required to synthesize this material.
In the last decade, a number of investigators have examined the effects of administering Cr(pic)3 (and in some cases other forms of chromium(III)) to rats on regular diets [30-33]. After an initial preliminary report which suggested beneficial effects on blood variables [30], detailed examinations of the effect of Cr(pic)3 administration in amounts up to 1500 μg/kg diet for up to 24 weeks have found no acute toxic effects [31-33]. However, the compound and other chromium sources examined (most notably “Cr nicotinate” and chromium chloride) also had no effect on body mass, percentage lean or fat content, tissue size (heart, testes, liver, kidney, muscle, epididymal fat, spleen, and kidney), or blood variables (fasting glucose, insulin, cholesterol, etc.). No differences in the gross histology of the liver or kidney (organs where chromium(III) preferentially accumulated) were found, although chromium did accumulate in these organs [33]. Another study compared the effects of a Cr-deficient diet with diets supplemented with ten different sources of chromium, including allowing rats to live in stainless steel cages. The Cr sources had no effect on body mass; all but one source decreased epididymal fat. Testes and liver masses tended to be lowered, whereas kidney, heart, and spleen masses were not significantly altered. Supplemental Cr had no effect on serum triglycerides or cholesterol, and only one source resulted in lower serum glucose [34]. While these studies did not manifest any acute toxicity, the lack of beneficial effects of Cr(pic)3 supplementation on growth, fat content or glucose, insulin, or cholesterol concentrations raises questions about its therapeutic potential. Recently the safety of intaking Cr(pic)3 has been questioned, especially in regards to its potential to cause clastogenic damage [35,36]. At physiologically-relevant concentrations of chromium (120 nM) and biological reductants such as ascorbic acid and thiols (5 mM), Cr(pic)3 has been shown to catalytically produce hydroxyl radicals which cleave DNA[35]. This ability stems from the combination of chromium and picolinate; the picolinate ligands prime the redox potential of the chromic center such that it is susceptible to reduction. The reduced chromium species interacts with dioxygen to produce reduced oxygen species including hydroxyl radical. These studies are in agreement with earlier studies which showed that mutagenic forms of chromium(III) required chelating ligands containing pyridine-type nitrogens coordinated to the metal [37].
Cr3 and other compounds of the general formula [Cr3O(O2CR)6L3]n+ (where M is a trivalent transition metal ion, R is an organic group, and L is a terminal ligand such as water or pyridine) are called basic chromium carboxylates. [For Cr3, R=Et, L=H2O, and n=1 Complex 1]. They historically have been prepared by “three general methods: reaction of freshly precipitated chromium(III) hydroxide with the carboxylic acid, either neat or in aqueous solution; reduction of chromium trioxide in the acid media, the reductant being the acid itself (acetic or formic), or some added substance such as ethanol; and oxidation of chromium(II) carboxylates.” [38] In terms of synthesizing Cr3 or any of these on an industrial scale, all of these avenues are problematic. Cr(II) compounds are air sensitive and would require specialized equipment. Chromium trioxide and other Cr(VI) complexes are carcinogenic; the possibility of incomplete reduction could represent a health risk. Freshly prepared chromic hydroxide is a very fine powder, which is very difficult to filter on a laboratory scale and should be extraordinarily difficult to filter to isolate on an industrial scale. Additionally, the filtrate, which could potentially be recycled to some degree, is an extremely caustic solution.
For Cr3 specifically, a few syntheses have been described in the literature. The first reported synthesis of Cr3 dates back to 1908 by A. Werner [39]; Werner started from chromic hydroxide, although the formula for the chloride salt of Cr3 was incorrectly reported as [Cr3(OH)2(propionate)6]Cl.5H2O, although this is remarkably close to the correct [Cr3O(propionate)6(H2O)3]Cl.(H2O)n, especially given the resources available at the time. Weinland and Hoehn apparently also made Cr3 from Cr(VI) in 1911 [40]. Another reported synthesis of Cr3 in English appeared in 1966 by Earnshaw and coworkers, although the cation was still incorrectly identified as [Cr3(OH)2(propionate)6]+ [41]. They basically used the chromic hydroxide procedure of Werner. The use of chromic hydroxide to generate Cr3 was also reported by Szymanska-Buzar and Ziolowski [42]; their procedure was a simple variation on that of Werner. Kapoor and Sharma [43] reported synthesizing Cr3 from anhydrous CrCl3 and propionic acid in CCl4, starting at −10° C. and then heating the reaction to reflux with the generation of HCl. The use of an organic solvent, especially a carcinogen, is less than ideal, as is the generation of HCl. Antsyshkina and coworkers [44] prepared Cr3 by heating a mixture of chromium nitrate and propionic anhydride; the mixture was heated until toxic, noxious fumes of nitrogen oxide, NOx, appeared. Finally, following the procedure developed by Vincent and coworkers for making basic chromium carboxylates in nonaqueous solvents [45], Fujihara, et al. [46] prepared Cr3 from chromium nitrate and propionic acid in acetone.
Thus, all synthetic procedures for preparing Cr3 to date involve the use of nonaqueous solvents, the formation difficult to isolate chromic hydroxide as an intermediate, or the production of toxic or hazardous byproducts. A one-pot, aqueous synthesis of Cr3 and related complexes without the production of toxic byproducts, which can be scaled from the laboratory to an industrial setting, is needed.